Potentiometric Electrode, Gradient Polymer, Uses And Method Of Preparation Therefor

ABSTRACT

The present invention relates to a potentiometric electrode and gradient polymer, both comprising electrically conducting particles, which increase in concentration away from one surface, ionophore molecules, which increase in concentration towards the same surface surface, and an electrical connection which passes proximal to said electrically conducting particles. The invention further relates to devices incorporating said electrode or gradient polymer, and to a method for their preparation. The new materials are highly robust and reliable.

FIELD OF THE INVENTION

The present invention relates to the field of potentiometric electrodes,gradient polymers and electrical or sensing components comprising saidgradients. It also relates to methods for preparing potentiometricelectrodes and gradient polymers.

BACKGROUND TO THE INVENTION

Potentiometric electrodes have widespread applications in the fields ofbiology, chemistry, and medicine. They are used for detecting andmeasuring the concentration of ion species in solution, the best knownexample being the pH-meter. The operating principles are well known.

Potentiometric electrodes can also be used as sensors to detectanalytes. Analyte may be any desired target that has a correspondingionophore that is capable of specifically interacting with targetsubstances of interest. Examples of analytes include organic acids,amines, amino alcohols and pharmaceutical drugs. Such electrodescombined with suitable ionophores are capable of detecting acorresponding analyte. The applications of potentiometric electrodes arenumerous, including biomedical research, clinical testing, obtainingdrug data, industrial pollution testing, food testing andchemical-process control.

At present, there is a strong drive towards application ofelectrochemical sensing of ionizable substances in miniaturizedtechniques, such as capillary electrophoresis, and microchip arrays.

Potentiometric electrodes coupled to separation methods (e.g. HighPerformance Liquid Chromatography, “HPLC”) are practically non-existing.However, such methods can give access to many interesting substanceswhich are available in minute quantities only, and often in admixturewith other substances. For these applications, sensors with lowspecificity are required, in contrast to batch techniques in which highspecificity is required.

Patent application EP 0 767 372 A1 discloses a substrate electrodecomprising an epoxy resin (or polyester-, or polyimide resin) cylindercontaining carbon fibers. A classical poly (vinylchloride) (PVC)-basedpotentiometric membrane is coated on this substrate electrode. Theauthors made a sophisticated design to optimize the membrane—substrateelectrode contact,

Patent application EP 0 684 466 A2 discloses a substrate electrode whichis a solid conductor or semiconductor. It is coated with a classicalPVC/plasticiser/ionophore mixture, plus an additional conductivepolymer.

Patent application EP 0 300 662 A2 discloses a conventional coated wirepotentiometric electrode which is optimized for the measurement ofcationic and anionic surfactants.

Patent application U.S. Pat. No. 5,552,032 describes a chloride (orgenerally, a halide ion) sensitive potentiometric electrode. It is avariant of the potentiometric sensors based on silver halide crystalelectrode materials.

A problem with potentiometric electrodes of the prior art is the lack ofstability (potential drift), mechanical robustness and sensitivity.Their life span is unpredictable, so requiring regular financialexpenditure in the form of maintenance on the part of the owner.Furthermore, they are unsuitable for use in HLPC detection and CapillaryElectrophoresis (CE) detection, dissolution testing of pharmaceuticals,wherein their behaviour is difficult to predict, and some designs cannotbe used with the ion-pairing agent frequently used in HPLC and CEseparations or with the detergents used in dissolution testing.

There is clearly a need for a new potentiometric electrode, suitable foruse in sensing analytes which overcomes the problems of the prior art.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a potentiometric electrodefor selective analyte detection in a sample comprising:

-   -   a sensing body made from polymeric material comprising:        -   electrically conducting particles, which increase in            concentration away from a sample contact surface,        -   ionophore molecules, which increase in concentration towards            the sample contact surface, and        -   an electrical connection which passes proximal to said            electrically conducting particles.

Another embodiment of the present invention is a potentiometricelectrode as described above wherein said electrical connection is madeof copper.

Another embodiment of the present invention is a potentiometricelectrode as described above wherein the polymeric material is polyvinylchloride.

Another embodiment of the present invention is a potentiometricelectrode as described above wherein the polymeric material comprisesone or more of poly(n-butylacrylate), poly(butylacrylate), cross-linkedpoly(butylacrylate), polycarbonate, polystyrene, polymethylmethacrylate,poly(vinylchloride-co-vinylacetate-co-vinylalcohol), polysiloxane, DC200 silicon oil, polyvinyl chloride, or high molecular weight polyvinylchloride.

Another embodiment of the present invention is a potentiometricelectrode as described above, wherein the maximum concentration ofelectrically conducting particles is 65% w/w.

Another embodiment of the present invention is a potentiometricelectrode as described above, wherein the electrically conductingparticles are graphite powder.

Another embodiment of the present invention is a potentiometricelectrode as described above wherein said electrically conductingparticles are any of carbon powder, graphite powder, synthetic graphitepowder with a diameter of 1 to 2 micrometer.

Another embodiment of the present invention is a potentiometricelectrode as described above, wherein said electrically conductingparticles are any of electropolymerised materials, oxidized polypyroleand its derivatives, oxidized polythiophenes, polyaniline, noble metals,gold or platinum.

Another embodiment of the present invention is a potentiometricelectrode as described above wherein the ionophore molecules are one ormore selected from the group consisting of compounds 1 to 10 in FIG. 11.

Another embodiment of the present invention is a potentiometricelectrode as described above said ionophore molecules are one or moreselected from the group consisting of tetra (p-chloro)phenylborate,methyltridodecylammoniumchloride and compounds 11 to 21 in FIG. 11.

Another embodiment of the present invention is a potentiometricelectrode as described above wherein the sensing body is cylindrical inshape.

Another embodiment of the present invention is a potentiometricelectrode as described above wherein the polymeric material is at leastpartly enclosed in a housing.

Another embodiment of the present invention is a potentiometricelectrode as described above wherein the ionophore molecules and theelectrically conducting particles mix only in a transition region.

Another embodiment of the present invention is a potentiometricelectrode as described above wherein the region of polymeric materialcomprising ionophore molecules, comprises more plasticiser than theremainder of the polymeric material.

Another embodiment of the present invention is a chromatographic flowcell comprising an potentiometric electrode as described above.

Another embodiment of the present invention is a chromatographic flowcell as described above, comprising a nozzle allowing eluent to besprayed onto a sample contact surface.

Another embodiment of the present invention is a method for making apotentiometric electrode as described above comprising the steps of:

-   -   (a) preparing a suspension of electrically conducting particles        and in a solution of polymeric material in a solvent,    -   (b) inserting an electrical conductor therein,    -   (c) drying the suspension, so forming a solid composite polymer        with electrically conducting particles therein,    -   (d) adding, to a surface of the composite of step (c) a solution        of polymeric material, electrically conducting particles and        ionophore molecules,    -   (e) drying the mixture,    -   (f) repeating steps (d) and (e), with decreasing concentrations        of electrically conducting particles, and increasing        concentrations of ionophore molecules, and    -   (g) obtaining a potentiometric electrode.

Another embodiment of the present invention is a method for making apotentiometric electrode as described above comprising the steps of:

(a) preparing a suspension of electrically conducting particles in asolution of polymer in a solvent,

(b) inserting an electrical conductor therein,

(c) drying the suspension, so forming a solid composite polymer withelectrically conducting particles therein,

(d1) adding, to a surface of the particle/polymer composite, a solutionof polymer and ionophore.

(e1) drying the mixture.

(f1) repeating steps (d1) and (e1), and

(g1) obtaining a potentiometric electrode with composite gradientproperties and no interfaces.

Another embodiment of the present invention is a method for making apotentiometric electrode as described above comprising the steps of:

(a1) preparing a suspension of electrically conducting particles in asolution of polymer in a solvent,

(b1) injecting the suspension into distilled water, so forming aprecipitate,

(b2) reducing the size of the precipitate to form a residue,

(c1) drying the residue by pressing to form a conductingparticle/polymer composite, followed by steps (d) to (g) or (d1) to (g1)as defined above.

Another embodiment of the present invention is a method as describedabove wherein the relative proportion (w/w) of said electricallyconducting particles to said polymer is in the range 20 to 90%electrically conducting particles.

Another embodiment of the present invention is a method as describedabove wherein said electrically conducting particles are any of carbonpowder, graphite powder, synthetic graphite powder with a diameter of 1to 2 micrometer.

Another embodiment of the present invention is a method as describedabove wherein said electrically conducting particles are any ofelectropolymerised materials, oxidized polypyrole and its derivatives,oxidized polythiophenes, polyaniline, noble metals, gold or platinum.

Another embodiment of the present invention is a method as describedabove wherein said polymer is any of poly(n-butylacrylate),poly(butylacrylate), polycarbonate, polystyrene, polymethylmethacrylate,poly(vinylchloride-co-vinylacetate-co-vinylalcohol), polysiloxane, DC200 silicone oil or high molecule weight polyvinyl chloride (PVC).

Another embodiment of the present invention is a method as describedabove wherein the proportion (w/v) of solid components (electricallyconducting particles and polymer), to solvent in the suspension of step(a) or (a1) is 1:(1 to 10).

Another embodiment of the present invention is a method as describedabove wherein the solvent of step (a) or (a1) is any of DMSO, 111trichloro ethane, CCl₄, N,N-dimethylformamide and N,N-dimethylacetamide,and THF.

Another embodiment of the present invention is a method as describedabove wherein the solvent of step (d) or (d1) is any of DMSO, 111trichloro ethane, CCl₄, N,N-dimethylformamide and N,N-dimethylacetamide,and THF.

Another embodiment of the present invention is a method as describedabove wherein said ionophore molecules are one or more selected from thegroup consisting of compounds 1 to 10 in FIG. 11.

Another embodiment of the present invention is a method as describedabove wherein said ionophore molecules are one or more selected from thegroup consisting of tetra(p-chloro)phenylborate,methyltridodecylammoniumchloride and compounds 11 to 21 in FIG. 11

Another embodiment of the present invention is a method as describedabove wherein said polymer in step (d) or (d1) is any which is in therubber phase at or below room temperature.

Another embodiment of the present invention is a method as describedabove wherein said polymer in step (d) or (d1) is poly(butylacrylate).

Another embodiment of the present invention is a method as describedabove wherein composition of the solid components (polymer andionophore) in step (d) or (d1) comprises 85 to 99% polymer.

Another embodiment of the present invention is a method as describedabove wherein the composition of the solid components (polymer andionophore) in step (d) or (d1), comprises 0.1 to 3% ionophore.

Another embodiment of the present invention is a method as describedabove wherein the ratio (w/v) of solid components (polymer andionophore) to solvent is 1:(8 to 12) in step (d) or (d1).

Another embodiment of the present invention is a method as describedabove wherein the solution of step (d) or (d1) further comprisesplasticiser.

Another embodiment of the present invention is a method as describedabove wherein the composition of the membrane components (plasticiser,polymer and ionophore) in step (d) or (d1) comprises 55 to 75%plasticiser.

Another embodiment of the present invention is a method as describedabove wherein said plasticiser is any of o-nitrophenyl octyl ether,dioctyl sebacate, bis(2-ethylhexyl)phtalate, tris(2-ethylhexyl)phosphateor tris(2-ethylhexyl)trimellitate.

Another embodiment of the present invention is a method as describedabove wherein the composition of the membrane components (plasticiser,polymer and ionophore) in step (d) or (d1), comprise 28 to 38% polymer.

Another embodiment of the present invention is a method as describedabove wherein the composition of the membrane components (plasticiser,polymer and ionophore) in step (d) or (d1), comprises 0.1 to 3%ionophore.

Another embodiment of the present invention is a method as describedabove wherein the ratio (w/v) of membrane components (plasticiser,polymer and ionophore) to solvent is 1:(8 to 12) in step (d) or (d1).

Another embodiment of the present invention is a method for making apotentiometric electrode as described comprising the steps of

a3) preparing a paste comprising:

-   -   a suspension of electrically conducting particles defined in        step (a) or step (a1) above, with or without solvent, or    -   a suspension of electrically conducting particles defined in        step (a) or step (a1) above, with or without solvent, in which        the polymer is polysiloxane, or    -   a suspension of electrically conducting particles defined in        step (a) or step (a1) above, with or without solvent, in which        the polymer is DC 200 silicon oil,        b3) inserting an electrical conductor therein,        c3) adding to a surface of the particle/polymer composite a        mixture comprising base, curing agent, and ionophore, optionally        dissolved in solvent.        d3) degassing the construction,        e3) heating the construction, and        f3) obtaining a potentiometric electrode with composite gradient        properties and no interfaces.

Another embodiment of the present invention is a method as describedabove wherein the solvent of step (a) is tetrahyrofuran.

Another embodiment of the present invention is a method as describedabove wherein the maximum concentration of electrically conductingparticles is 20 to 90% w/w.

Another embodiment of the present invention is a method as describedabove wherein the maximum concentration of ionophore molecules is 0.1 to5% w/w.

Another embodiment of the present invention is a gradient polymercomprising:

-   -   electrically conducting particles, which increase in        concentration away from a surface,    -   ionophore molecules, which increase in concentration towards the        surface.

Another embodiment of the present invention is a gradient polymersubstantially formed from polymeric material comprising two surfaces,said polymeric material comprising:

-   -   electrically conducting particles which decrease in        concentration away from one surface,    -   ionophore molecules decease in concentration away from the other        surface.

Another embodiment of the present invention is a gradient polymersubstantially formed from polymeric material comprising two surfaces,said polymeric material comprising electrically conducting particles,which decrease in concentration away from both surfaces.

Another embodiment of the present invention is a gradient polymer asdescribed above, wherein the polymer comprises one or more of thepolymers defined above.

Another embodiment of the present invention is a gradient polymer asdescribed above, wherein the polymer comprises one or more of theelectrically conducting particles defined above.

Another embodiment of the present invention is a gradient polymer asdescribed above, wherein the polymer comprises one or more of theionophores defined above.

Another embodiment of the present invention is a gradient polymer asdescribed above, comprising the steps defined in the methods above,wherein one or more additional electrical conductors are incorporated.

Another embodiment of the present invention is a method for preparing agradient polymer as described above comprising the steps defined in themethods above, wherein the ionophore of step (d) and (d1) is absent.

Another embodiment of the present invention is a method for preparing agradient polymer comprising the steps defined in the methods above,further comprising the step of joining two gradient polymers together atthe surfaces of lowest concentration of electrically conductingparticles.

Another embodiment of the present invention is a battery comprisinggradient polymer as described above.

Another embodiment of the present invention is a variable resistorgradient polymer as described above.

Another embodiment of the present invention is a variable capacitorgradient polymer as described above.

Another embodiment of the present invention is a pressure sensorgradient polymer as described above.

Another embodiment of the present invention is a solvent or lipophilicmolecule sensor gradient polymer as described above.

Another embodiment of the present invention is a use of a gradientpolymer as described above for sensing analytes.

Another embodiment of the present invention is a use of a potentiometricelectrode as described above for inline monitoring the dissolution of adrug from a drug formulation.

FIGURES

FIG. 1. An example of a potentiometric electrode according to theinvention.

FIG. 2. An example of a potentiometric electrode according to theinvention.

FIG. 3. Examples (a, b and c) of continuous gradient polymers with noisedue to uneven distribution and particle size.

FIG. 4. Examples (a, b and c) of discontinuous gradient polymers.

FIG. 5. Gradient of components within a gradient polymer of an electrodefrom isolator (left) to membrane (right), wherein A is PVC, B is DOS andC is ionophore and ions.

FIG. 6. Gradient of components within a gradient polymer of an electrodefrom conducting composite (left) to membrane (right), wherein A isgraphite, B is PVC, C is DOS and D is ionophore and ions.

FIG. 7. Photograph of a section of the electrode in reflectionmicroscopy.

FIG. 8. Drift of an electrode in a 30 mg/l solution of dapoxetine at 37deg C. over approximately 7 days.

FIG. 9. The graphite concentration in a gradient polymer of two-sidedsensor.

FIGS. 10A and 10B. An example of a flow cell according to the invention.

FIG. 11. Examples of suitable ionophores according to the invention,including compound 11: calix[6]arene, compound 12: calix[4]arene,compound 13: valinomycin, compound 14: nonactin, compound 15: amineionofore 1, compound 16: dibenzo-18-crown-6, compound 17:Dibenzo-24-crown-8, compound 18: Dibenzo-30 crown-10, compound 19:cyclodextrin, compound 20: phosphoryl derivate, and compound 21 (a crownether).

FIG. 12. HPLC traces obtained using an electrode according to theinvention based on compound 3. The upper trace shows a chromatogram forthe mixture of acids in Table 1. The lower trace presents a chromatogramof an injected beer sample.

FIG. 13. An HPLC chromatogram recorded with an electrode based oncompound 9, with the mixture of acids according to Table 1 as sample.

FIG. 14. HPLC chromatograms (A and B) recorded with an electrode basedon compound 10, with the mixture of acids according to Table 1 assample.

FIG. 15. Calibration curves obtained for three acids in HPLC conditions,with an electrode comprising compound 7.

FIG. 16. Curve demonstrating the logarithmic response of an electrode ofthe invention to concentration in batch conditions.

FIG. 17. The monitoring of dissolution of galantamine in HCl using apotentiometric electrode of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a potentiometric electrode fordetecting analytes in a sample comprising

-   -   a sensing body made substantially from polymeric material        comprising:        -   electrically conducting particles, which increase in            concentration away from a sample contact surface,        -   ionophore molecules, which increase in concentration towards            the sample contact surface, and        -   an electrical connection which passes proximal to said            electrically conducting particles.

Thus, one embodiment of the present invention is an analyte-selectivepotentiometric electrode having a sample contact surface, said electrodebeing substantially formed from polymeric material, said polymericmaterial comprising:

-   -   electrically conducting particles, which increase in        concentration away from the sample contact surface,    -   ionophore molecules capable of binding to said analyte, which        increase in concentration towards the sample contact surface,        and    -   an electrical connection which passes proximal to said        electrically conducting particles.

Another embodiment of the present invention is a gradient polymerpresent in an electrode of the present invention.

According to one embodiment of the present invention a gradient polymeris substantially formed from polymeric material comprising two surfaces,said polymeric material comprising:

-   -   electrically conducting particles, which decrease in        concentration away from one surface,    -   ionophore molecules which decrease in concentration away from        the other surface,    -   optionally an electrical connection which passes proximal to        said electrically conducting particles.

According to one embodiment of the present invention a gradient polymeris substantially formed from polymeric material comprising two surfaces,said polymeric material comprising:

-   -   electrically conducting particles, which decrease in        concentration away from both surfaces,    -   optionally electrical connection(s) which passes proximal to        said electrically conducting particles.

An electrode or gradient polymer according to the invention, whichcomprises polymeric material throughout has been found by the inventorsto be extremely mechanically robust and sensitive. Furthermore, such anelectrode or gradient polymer is suitable for use in HPLC, CE andpharmaceutical applications such as dissolution testing and retains itssensitivity during very long operations. In the area of dissolutiontesting, the inventors have found that the robust potentiometricelectrode performs more reliably than the presently widely usedspectroscopic UV fiber-optics sensors. The electrode or gradient polymerdoes not have a separate and distinct membrane comprising ionophoremolecules. Instead, the part of the electrode or gradient polymercomprising ionophore molecules is part of the polymeric material of thebody, and the electrode or gradient polymer is one piece. Furthermore,the electrode or gradient polymer is more robust and the inventors havefound it is free of abrupt changes in potential which can occur atunpredictable intervals with conventional “coated-wire” electrodes. Whenit is described that the concentration of a ionophore or electricallyconducting particle increases or decreases, it means such change is notabrupt but is gradual or progressive. As elaborated below, the change inconcentration of ionophore or electrically conducting particle is agradient over distance in respect of the longitudinal body or in respectof the sample contact surface. It is mechanically more stable becausethere is no abrupt interface between the ionically conducting part andthe electronically conducting part.

The sensing body of the electrode comprises the gradient polymer, saidgradient having a variety of applications including use in a flow cell,a dissolution sensor, battery, variable resistor, variable capacitor,pressure sensor and solvent or lipophilic molecular sensor.

One embodiment of the present invention is an electrode or gradientpolymer as disclosed herein wherein the electrical connection is made ofany conducting metal such as silver, platinum, gold, aluminium.

Another embodiment the present invention is an electrode or gradientpolymer as disclosed herein wherein the electrical connection is made ofcopper. The use of copper provides good conductivity and is inexpensive.Because an electrode or gradient polymer of the invention can avoid theuse of precious metal such as gold, silver or platinum, said electrodeprovides for a very low cost device.

Another embodiment of the present invention is an electrode or gradientpolymer as disclosed herein, wherein polymeric material is any polymericmaterial which is in the rubber state, or which is brought into thisstate by the addition of plasticiser. Examples of such materials includepoly(n-butylacrylate), poly(butylacrylate), cross-linkedpoly(butylacrylate), polycarbonate, polystyrene, polymethylmethacrylate,poly(vinylchloride-co-vinylacetate-co-vinylalcohol), polysiloxane, DC200 silicon oil, polyvinyl chloride (PVC), or high molecular weight PVCor a combination of two or more thereof.

Another embodiment of the present invention is an electrode or gradientpolymer as disclosed herein, wherein polymer is polyvinyl chloride(PVC). The inventors have found that PVC provides good strength and iscompatible with the electrically conducting particles and ionophores ofthe electrode or polymer gradient.

Another embodiment of the present invention is an electrode or gradientpolymer as disclosed herein, wherein the polymer comprisespoly(butylacrylate). The electrode may comprise poly(butylacrylate), orpoly(butylacrylate) in combination with PVC. The inventors have foundthat provides good adhesive properties for the construction of theelectrode and is compatible with the electrically conducting particlesand ionophores of the electrode.

The region of polymeric material comprising ionophore molecules (FIGS.1, 11) is also known herein the membrane, while the remainder of thepolymeric material (FIGS. 1, 10), which comprises electricallyconducting particles is also known as the conducting composite.

According to one aspect of the invention, the membrane region comprisesmore plasticiser than the conducting composite region. According to thisaspect of the invention, the amount of plasticiser in the membraneregion is between 40 to 80%, 50 to 70%, 60 to 70%, 60 to 75% and ispreferably 66%. According to this aspect of the invention, the amount ofplasticiser in the conducting composite region is between 0 to 37%, 0 to20%, 0 to 10%, and is preferably 0%. According to this aspect of theinvention, the plasticiser is any of o-nitrophenyl octyl ether (o-NPOE),dioctyl sebacate (DOS), bis(2-ethylhexyl)phtalate (DOP),tris(2-ethylhexyl)phosphate (TOP) or tris(2-ethylhexyl)trimellitate(TOTM).

According to another aspect of the invention, the membrane regioncomprises the same polymer as in the conducting composite region. Forexample, the membrane region and the conducting composite region mayboth comprise PVC. Alternatively, both regions may comprisepoly(butylacrylate).

According to another aspect of the invention, the membrane regioncomprises one or more polymers different from the conducting compositeregion. For example, the membrane region may comprisepoly(butylacrylate), and the conducting composite region may comprisePVC. Alternatively, the membrane region may comprise a mixture ofpoly(butylacrylate) and PVC, and the conducting composite region maycomprise poly(butylacrylate). Alternatives to poly(butylacrylate) may beemployed such as any polymer which has a low glass transitiontemperature (low Tg) i.e. is in the rubber phase at or below roomtemperature (below 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20 deg C.).

The use of poly(n-butylacrylate) (low Tg) in the conducting compositeregion provides an ideal self-gluing base for the mounting of theionophore region.

As used herein for substance amounts, percentages refer to w/w unlessotherwise stated.

Another embodiment of the present invention is an electrode or gradientpolymer as disclosed herein, wherein the concentration of electricallyconducting particles is a gradient across the longitudinal body of theelectrode or gradient polymer. The concentration of electricallyconducting particles is highest towards one or both surfaces. One aspectof the invention is an electrode or gradient polymer as disclosedherein, wherein the maximum concentration of electrically conductingparticles in the conducting composite region is equal to or more than20, 30, 40, 50, 60, 70, 80 or 90% electrically conducting particles, ora value in the range between any of the two aforementioned percentages.It may be 20 to 90%, 50 to 80%, 60 to 80%, 70 to 80%, 50 to 70%, 60 to70%, 50 to 60% and is preferably equal to or more than 65% electricallyconducting particles.

By proximal in reference to the electrical connection as used hereinmeans the electrical connection passes through a zone of electricallyconducting particles. It is an aspect of the invention that theelectrical connection passes through a zone of maximum concentration ofelectrically conducting particles.

The gradient of electrically conducting particles provides a continuoustransition between ionic conductivity (low particle concentration) toelectronic conductivity (high particle concentration)

Another embodiment of the present invention is an electrode or gradientpolymer as disclosed herein, wherein the minimum concentration ofelectrically conducting particles in the membrane region is equal to orless than 5, 10, 20, 40 or 40% or a value in the range between any ofthe two aforementioned percentages electrically conducting particles. Itmay be 0 to 40%, 0 to 30%, 0 to 20%, 0 to 10%, 0 to 5%, 0 to 3%electrically conducting particles, and is preferably 0%.

Another embodiment of the present invention is an electrode or gradientpolymer as disclosed herein, wherein the electrically conductingparticles are carbon powder, preferably, graphite powder, even morepreferably synthetic, with a diameter of 1-2 micrometer. Otherelectrically conducting particles suitable for use according to theinvention include, but are not limited to electropolymerised materials(or any other ion electron converter) such as oxidized polypyrole andits derivatives, oxidized polythiophenes and polyaniline, noble metals,gold or platinum.

Another embodiment of the present invention is an electrode or gradientpolymer as disclosed herein, wherein the electrically conductingparticles are of one type (e.g. all graphite powder). Another embodimentof the present invention is an electrode as disclosed herein, whereinthe electrically conducting particles are of more than one type. Forexample, an electrode may comprise electrically conducting particles ofgraphite powder and gold.

Another embodiment of the present invention is an electrode or gradientpolymer as disclosed herein, wherein an ionophore molecule is anymolecule suitable for non-covalent binding to the analyte of interest.According to one embodiment of the invention, an ionophore specificallybinds to an analyte. According to one embodiment of the invention, anionophore specifically binds to a group of related analytes. Byrecognizing one analyte, or a groups of analytes, an ionophore accordingto the invention enables the specific qualitative and/or quantitativedetection of said analytes in the presence of other substances.

An analyte as used herein is the substance of interest which isdetectable using an electrode or gradient polymer of the invention. Tobe useful in the invention, the analyte is ionizable i.e. it can formcations and/or anions. For example, an analyte may be a cation presentin a liquid, in a gel, on a surface, in a sample etc. According to theinvention, an analyte or a family of related analytes binds specificallyto an ionophore present in an electrode or gradient polymer of theinvention.

A sample according to the invention is any in which an analyte iscapable of being present i.e., in which an analyte is ionisable. Asample may take the form of, for example, a liquid, a vapour, a gel, avapour, a semi-solid, a liquid film, a moisture film, a suspension, acolloid, an heterogenous mixture, an homologous mixture, etc. Examplesof samples include, but are not limited to food-stuffs, drinks, bodilyfluids, drinking water, waste water, environmental specimens,chemical-production samples, chemical samples, biological samples etc.

According to one aspect of the invention, the analyte is present as anion pair in the electrode or gradient polymer, particularly in membraneregion.

Ionophores may be any of the art, including tetra(p-chloro)phenylborate(TCPB) for cationic analytes, and methyltridodecylammoniumchloride(MTDDACl) for anionic analytes. Other ionophores are any molecularrecognition compound which can be made soluble in the lipophilicelectrode material or gradient polymer, and insoluble in a hydrophilicsample. Examples synthesized by applicants for the determination oforganic acids are given in FIG. 11. Other ionophores known in the artsuitable for use in the present invention include those mentioned inBühlmann et al. Chemical Reviews 1998 Vol. 98 No 4 p 1650-1687 which isincorporated herein by reference. Other ionophores known in the artsuitable for use in the present invention include calixarenes (e.g. FIG.11 compounds 11, 12 and 15) crown ethers (e.g. FIG. 11 compounds 16, 17,18 and 21) cyclodextrins (e.g. FIG. 11 compound 19) phosphoryl derivates(e.g. FIG. 11 compound 20) and antibiotics (e.g. FIG. 11 compounds 13and 14). Improved solubility characteristics may be obtained byincorporating a lipophilic “tail” into the molecular recognitioncompound.

The sensitivity and selectivity of potentiometric electrodes of the artfor the determination of organic ionizable analytes is largelydetermined by the lipophilicity of the analyte. Best results areobtained with analytes having large positive log P values, wherein P isthe distribution coefficient of the analyte over a n-octanol/water twophase system. Most organic acids from the food and drinks industry havelow (negative) log P values, and will, therefore, be difficult targets.The inventors have found that a combination of the electrode or gradientpolymer as described herein, and a group of urea- or azamacrocycle-basedionophores as shown in FIG. 11 allows the detection of such lipophilicanalytes, thereby enabling important analyses to be performed in batchand in HPLC modes.

According to one aspect of the invention, an ionophore is based on aureum derivative or on an azamacrocycle, and is any selected from thegroup consisting of compounds 1 to 10 in FIG. 11.

Another embodiment of the present invention is an electrode or gradientpolymer as disclosed herein, wherein the concentration of ionophore is agradient across the longitudinal body of the electrode or gradientpolymer. The concentration of ionophore is highest towards one surfaceswhen a single gradient is present. When two gradient polymers aredisposed ‘back to back’ as mentioned below, the concentration ionophoreis highest towards the midpopint. One aspect of the invention is anelectrode or gradient polymer as disclosed herein, wherein the maximumconcentration of ionophore in the membrane region is 0.1 to 20%, 0.1 to10%, 0.1 to 9%, 0.1 to 8%, 0.1 to 7%, 0.1 to 6%, 0.1 to 5%, 0.1 to 4%,0.1 to 3%, 0.1 to 2%, 0.1 to 1%, and is preferably 1%, most preferably 1to 2% ionophore. For the determination of organic acids, this ionophoreis any one of the molecules from FIG. 11. These surface-active receptormolecules largely increase the detectability for organic acids. MTDDAClis added in a 0.2% ratio to obtain good ionic conductivity. Anotherembodiment of the present invention is an electrode or gradient polymeras disclosed herein, where the ionophores comprise a single ionophore(e.g. the ionophores are all compound 1 of FIG. 11). Another embodimentof the present invention is an electrode as disclosed herein, whereinthe ionophores are an heterogenous mix of at least two ionophores. Forexample, an electrode may comprise a mixture of compound 1 and 2 shownin FIG. 11.

Another embodiment of the present invention is an electrode or gradientpolymer as disclosed herein, wherein the minimum concentration ofionophore in the membrane region is 0 to 0.09%, 0 to 0.08%, 0 to 0.07%,0 to 0.05%, 0 to 0.025%, and is preferably 0% ionophore.

Another embodiment of the present invention is an electrode or gradientpolymer as disclosed herein, wherein the electrical connection does notpass through a zone of ionophore particles.

In another embodiment of the invention, the ionophore molecules and theelectrically conducting particles mix in a transition region between thezone of minimum concentration of ionophore molecules and the zone ofminimum concentration of electrically conducting particles.

According to an aspect of the invention, the transition region has adepth of less than or equal to 10, 50, 100, 200, 300, 400, 500, 600,700, 800, 900, 1000 or 3000 micrometers, or a value in the range betweenany two of the aforementioned depths. The depths may be in the range of10 to 1000 micrometer, 50 to 500 micrometer, 100 to 400 micrometers, 200to 300, 100 to 200 and 50 to 100 micrometers and preferably less than orequal to 200 micrometers.

According to another aspect of the invention, the nearest boundary ofthe transition region is located between 1 and 400, 10 and 400micrometer, 50 and 300 micrometers, 70 and 200 micrometers andpreferably 100 micrometers from the sample contact surface.

In this transition region, a conversion of ionic-to-electronicconductivity takes place more efficiently. According to another aspectof the invention, the depth of the transition region and/or the distanceof its boundary from the sample contact surface is such thationic-to-electronic conductivity takes place efficiently.

The gradient polymer and electrode as described herein, comprises agradient of electrically conducting particles and ionophores present inthe electrode forms a continuous transition from one functional layer toanother. This transition can be of any type including linear,exponential or curved as shown in FIGS. 3 a, 3 b and 3 c respectively.The gradient may be continuous. Alternatively, the gradient may not becompletely continuous, but may progress in discrete steps (FIG. 4) whichfollow a linear (FIG. 4 a), exponential (FIG. 4 b) or S-profile (FIG. 4a), for example. The gradient may be symmetrical or non-symmetrical(e.g. FIG. 5 shows a non symmetrical gradient).

The gradient between the conducting composite and the membrane secures amechanically strong adherence of the membrane to the conductor. Thegradient from the pure composite to the pure membrane spans typically200 micrometers, but it can vary from 10 to 1000 micrometers. Theinventors have observed that branches of composite (graphite/PVC) canextend into the membrane (PVC, DOS and ions). Isolated particles ofgraphite extend far into the membrane even in regions near to thesurface. Due to the extreme low concentration of these particles inthese regions, they do not reduce dramatically the impedance of theelectrode. A microscopy photograph of the membrane isolator gradient isshown in FIG. 7. From this, the inventors deduced a concentrationdistribution in the gradient shown in FIG. 6. For this distribution alinear variation of concentration was presumed, however the otherpresented models could also apply.

Within an electrode or gradient polymer of the present invention, othergradients may be present depending on the material used to make theisolator. Besides the gradient between conducting composite and agradient membrane, there may be a gradient between the isolator and themembrane and a gradient between the isolator and the conductor. For eachof these gradients there may be different gradient profiles for eachcomponent.

The gradient between the isolator and the membrane secures amechanically strong adherence of the membrane to the insulator.Furthermore this gradient has a higher impedance than the membrane andprevents any short-circuit. The gradient from the pure isolator to thepure membrane spans typically 200 micrometers, but it can vary from 10to 1000 micrometers. From the microscopy photograph of the membraneisolator gradient in FIG. 7 we deduced a concentration distribution inthe gradient shown in FIG. 5. For this distribution a linear variationof concentration was presumed, however the other presented models couldalso apply.

The gradient between the isolator and conducting composite is similar tothe previous gradients, and is an unintended result of the electrodeproduction as described above. The gradient may evolve from a graphiteconcentration equal to or greater than 90, 80, 70, 60, 50, 40, 30, or20% (or a value in the range between any of the two aforementionedvalues) to a concentration of 0% in the isolator. The concentration ofPVC may evolve from a concentration equal to or less than 10, 20, 30,40, 50, 60, 70, or 80% (or a value in the range between any of the twoaforementioned values) to a concentration equal to or less than 90, 95,or 100% in the isolator. Preferably, the concentration gradient evolvesfrom 60% graphite 40% PVC to 0% graphite 100% PVC. The length of thisgradient is smaller, and can vary from 1 to 100 micrometers but can belarger.

In another embodiment of the present invention, the electrode orgradient polymer further comprises a housing (also known herein as anisolator). The housing encloses at least part of a longitudinal body ofthe polymeric material. The housing leaves the sample contact surface atleast partly exposed. Said housing may serve a number of purposes, forexample, one or more of the following:

-   -   to physically protect the sensing portion when present in an        electrode,    -   to provide a reaching means for the sensing portion when        incorporated into an electrode,    -   to insulate the electrical connection.

The housing may be constructed from any insulating material such asglass, PVC, polycarbonate, polypropylene etc. It is preferably made ofPVC. The use of a PVC housing and solvent during preparation of theelectrode leads to an isolator-membrane gradient and a isolatorconductor composite gradient as described above, owning a solublisationof part of the housing by the solvent.

Another embodiment of the present invention is an electrode or gradientpolymer as disclosed herein, wherein the housing is a cylindricaljacket.

In another embodiment of the present invention, the polymeric materialis cylindrical in shape.

A cylindrical shape of the electrode or gradient polymer allows anelectrode so formed to have an sample contact surface which is circular,so matching the shape of commonly available containers such as microwell plates. Furthermore, this allows the housing of the electrode to becylindrical, permitting an efficient strength/weight use of housingmaterials.

In another embodiment of the present invention, the sample contactsurface of an electrode, or one or both said surfaces of gradientpolymer is circular. The may be between 0.1 and 5 mm, 0.1 and 4 mm, 0.1and 3 mm, 0.1 and 2 mm, and preferably 3 mm.

The inventors have found that an electrode or gradient polymer of thepresent invention with the lack of an interface provides a mechanicallymore robust construction which unexpectedly, has enhanced thesensitivity and conductivity properties which are more predictable indaily use.

Another embodiment of the present invention is a chromatographic flowcell comprising an electrode or gradient polymer of the presentinvention.

Another embodiment of the present invention is a dissolution electrodecomprising an electrode or gradient polymer of the present invention.The inventors have found that a potentiometric electrode made accordingto the present electrode has a very low drift, and which is predictable(FIG. 8). These properties allow measurements to be made inline—i.e. theelectrode registers continuously and in real time the concentrationchanges of the dissolution media; the electrode does not leave thedissolution media during dissolution. This contrasts to potentiometricelectrodes of the prior art in which samples are taken out and measured,or the electrode is intermittently introduced into the dissolutionsolution.

Besides the normal calibration curves, additional calibration techniquescan be applied to the present electrode or gradient polymer. Themeasurement of the end concentration is very sensitive to drift; thisproblem can be suppressed by single point calibration or standardaddition. Single point calibration usually performed soon aftercompletion of the dissolution by placing the electrode in a knownconcentration of analyte. This enables the correction of the calibrationvalues for the occurred drift by simple subtraction and interpolation. Aknown amount of analyte may also be added after completion of thedissolution (standard addition). Since the increase in voltage isproportional to the start concentration and the amount added, the endconcentration can be deduced. This also enables the calibration valuesto be corrected for the occurred drift by simple subtraction andinterpolation.

Another embodiment of the present invention is a chromatographic flowcell as disclosed herein, further comprising a means to spray eluentagainst the electrode or gradient polymer surface. Said configuration isa so called “walljet”.

The inventors have found that a flow cell according to the inventionobviates the main problem with classical flow-cells which is theunpredictable occurrence of high internal resistances. These come fromair bubbles sticking to the working electrode, or from referenceelectrodes with clogged membranes. Both causes are difficult to detectin classical flow cells. Classical cells have to disassembled, and eventhen, the cause of misfunction is not evident. Furthermore,re-assembling the flow-cell will generally not remove the problem. Thelatter phenomena make present electrochemical detection impractical forusers. The inventors have found that no such phenomena occur with theflow-cell of the present invention. A flow cell according to theinvention operates at zero pressure, allows immediate visual inspectionof air bubble formation at the electrode, and convenient and effectiveremoval of such in seconds. The reference electrode can also be removedin seconds time. The construction of the flow-cell is such that anindustrial standard reference electrode can be plugged in—no speciallyconstructed reference electrode is needed.

The gradient polymer described herein may comprise two surfaces and alongitudinal body. A cylinder, for example, comprising two circular endsand a longitudinal body is a typical shape of construction of gradientpolymer. The gradient polymer as already described above may compriseelectrically conducting particles increasing in concentration towardsone surface, and ionophore molecules increasing in concentration towardsthe other surface. A gradient polymer may also comprise two surfaces inwhich electrically conducting particles decrease in concentrationtowards the midpoint of both surfaces.

The gradient polymer can be implemented into a device wherever atransition between ionic and electrical conductivity is needed. Agradient polymer may thus be implemented, for example, into batteries.

It may also be implemented as a variable resistor or variable capacitor.In this construction the component is essentially two gradients ‘back toback’, in which electrically conducting particles decrease inconcentration towards the midpoint of both surfaces. The end surfaces ofthe components comprise a maximum concentration of electricallyconducting particles, which reduce in concentration towards the centre(midpoint) of the component. For example, the gradient may run from oneend from 60% graphite to 0% graphite in the centre and increases back to60% at the other end, this profile being shown in FIG. 9). The centralarea may be in the rubber phase at or below room temperature and maycontain ions and/or ionophores.

The gradient polymer may also be implemented into a pressure sensor. Forsuch sensor, again two ‘back to back’ gradients are formed in whichelectrically conducting particles decrease in concentration towards themidpoint of both surfaces. For example, the gradient may run at one endfrom 40% PVC 60% graphite to 66% DOS, 44% PVC, 0% graphite in the centre(midpoint) to 40% PVC 60% graphite at the other end. When this gradientis compressed, the capacitance and conductivity varies in proportion tothe pressure applied. Hence, a capacitance and/or impedance basedpressure sensor is obtained. Ionphores may be present or absent in thecentral area.

The gradient polymer as described in the above embodiments may also byimplement into a solvent or lipophilic molecular sensor. Such sensorbased on the swelling properties of the gradient. Many solvents becometrapped in the gradient polymer and cause swelling of the rubber phase.This characteristic affects the conductivity and capacitance of thesensor. Both properties can be used to determine the solventconcentration. The same system can be used for measurement of detergentsand molecules with high log P (>1). These molecules can be in thesolution or gas phase.

Method of Preparation

The potentiometric electrodes and gradient polymers can be preparedusing the methods described below. The methods are directed towards theconstruction of the electrode, however, the essential steps aredescribed for forming the gradient polymer. The skilled person may adaptthe described methods within his skill to prepare the gradient polymers.

Another embodiment of the present invention is a method for making apotentiometric electrode as described herein comprising the steps of:

(a) preparing a suspension of electrically conducting particles in asolution of polymer in a solvent,

(b) inserting an electrical conductor therein,

(c) drying the suspension, so forming a solid composite polymer withelectrically conducting particles therein,

(d) adding, to a surface of the particle/polymer composite, a solutionof polymer, electrically conducting particles, and ionophore.

(e) drying the mixture.

(f) repeating steps (d) and (e), with decreasing concentrations ofelectrically conducting particles, and increasing concentrations ofionophore, and

(g) obtaining a potentiometric electrode with composite gradientproperties and no interfaces.

Another embodiment of the present invention is a method for making apotentiometric electrode as described herein comprising the steps of:

(a) preparing a suspension of electrically conducting particles in asolution of polymer in a solvent,

(b) inserting an electrical conductor therein,

(c) drying the suspension, so forming a solid composite polymer withelectrically conducting particles therein,

(d1) adding, to a surface of the particle/polymer composite, a solutionof polymer and ionophore.

(e1) drying the mixture.

(f1) repeating steps (d1) and (e1), and

(g1) obtaining a potentiometric electrode with composite gradientproperties and no interfaces.

Another embodiment of the present invention is a method for making apotentiometric electrode as described herein comprising the steps of:

(a1) preparing a suspension of electrically conducting particles in asolution of polymer in a solvent,

(b1) injecting the suspension into distilled water, so forming aprecipitate,

(b2) reducing the size of the precipitate to form a residue,

(c1) drying the residue by pressing to form a conductingparticle/polymer composite, followed by steps (d) to (g) or (d1) to (g1)mentioned above

After hydration, the electrode is ready to be used.

The potentiometric electrode formed by the method is constructed from acontinuous polymer throughout the electrode, so providing the electrodewith an unexpected durability, toughness and sensitivity. The electrodealso has no interfaces, and so its enhanced sensitivity and conductivityproperties are more predictable in daily use

The properties and variations of the polymeric material, ionophore,electrically conducting particles and electrical conductors are the samefor the method as for the electrode or gradient polymer discussed above.

One embodiment of the invention is a method as disclosed herein, whereinthe suspension of step (a) is poured into a cylindrical mold. Thisallows the electrode so formed to have an sample contact surface whichis circular, so matching the shape of commonly available containers suchas micro well plates. Furthermore, this allows the housing of theelectrode to be cylindrical, permitting an efficient strength/weight useof housing materials. According to an aspect of the invention, the moldmay be the housing of the electrode.

The suspension of step (a) may be provided to the mold in one singleapplication, or by consecutive rounds of applying and drying until thedesired amount of suspension has been added. The drying may be performedat room temperature or elevated temperature (e.g. higher than 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90 deg C., or a valuein the range between any of the two aforementioned temperatures). Afterthe desired amount of composite has been added and the suspension dried,the surplus composite may be removed by grinding and polishing.

According to the present invention, the suspension of step (a) or (a1)comprises electrically conducting particles (e.g. graphite) and polymerin solvent. The relative proportion (w/w) of electrically conductingparticles to polymer is 20, 30, 40, 50, 60, 70, 80, 90% graphite, or avalue in the range between any of the two aforementioned percentages,the remainder being made to 100% with the appropriate mass of polymer.The preferred proportion of electrically conducting particles is 50 to70%, more preferably 55 to 65%, most preferably 60%, the remainder beingmade to 100% with polymer.

Similarly, the relative proportion (w/w) of polymer to electricallyconducting particles in the suspension of step (a) or (a1) is 10, 20,30, 40, 50, 60, 70, 80, % graphite, or a value in the range between anyof the two aforementioned percentages. The preferred proportion ofpolymer is 20 to 50%, more preferably 30 to 40%.

Examples of suitable polymers include poly(n-butylacrylate),poly(butylacrylate), polycarbonate, polystyrene, polymethylmethacrylate,poly(vinylchloride-co-vinylacetate-co-vinylalcohol), polysiloxane, DC200 silicone oil and preferably high molecule weight polyvinyl chloride(PVC).

The proportion of solid to solvent in the solution of step (a1) may beone part solid components (electrically conducting particles+polymer) toless than 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60 parts solvent, or avalue in the range between any of the two aforementioned values. Thepreferred ratio (w/v) of solid components (electrically conductingparticles+polymer) to solvent is 1:(1 to 10), more preferably 1:(1 to5), most preferably about 1:3.

The solvent used in the method of the present invention is any thatdissolves the polymer. Such solvents are known in the art, and includesDMSO, 111 trichloroethane, CCl₄, N,N-dimethylformamide andN,N-dimethylacetamide, and THF.

Another embodiment of the invention is a method as disclosed herein,wherein the solution of step (d) or (d1) (and (f)) further comprisesplasticiser. According to a method of the invention, the amount ofplasticiser in the polymeric material of the ionophore-containing regionis between 40 to 80%, 50 to 70%, 60 to 70%, 60 to 75% and is preferably66%. 50 to 75%, 55 to 75%, 60 to 75%, 65 to 75%, 50 to 70%, 50 to 65%,50 to 60 and preferably 66% plasticiser. According to this aspect of theinvention, the amount of plasticiser in the remainder of the polymericmaterial (conducting composite) is between 0 to 37%, 0 to 20%, 0 to 10%,and is preferably 0%.

Another embodiment of the invention is a method as disclosed herein,wherein the solution of step (d) or (d1) (and (f)) further comprisesionophore. According to a method of the invention, the maximumconcentration of ionophore in the electrode is equal to or less than0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9 or 10% w/w inthe membrane region. It may be between 0.1 and 10%, 0.1 and 9%, 0.1 and8%, 0.1 and 7%, 0.1 and 6%, 0.1 and 5%, 0.1 and 4%, 0.1 and 3%, 0.1 and2%, 0.1 and 1%, and is preferably less than or equal to 1% ionophore.

Another embodiment of the invention is a method as disclosed herein,wherein the solution of step (d) or (d1) and (f) further comprisespolymer. According to a method of the invention, the maximumconcentration of polymer in the membrane region of the electrode isbetween 20 and 45%, 20 and 40%, 20 and 35%, 20 and 30%, 25 and 45%, 30and 45%, 35 and 45%, and is preferably 33%.

According present method, the solution of step (d1) may comprise polymerand ionophore and no electrically conducting particles. Said solution isapplied to the composite of step (c) or (c1) by consecutive applications(step (f1)). The inventors have found that a gradient of ionophoreparticles increasing in the direction towards the sample contactsurface, and a gradient of electrically conducting particles decreasingin concentration in the direction towards the sample contact surface issurprisingly formed, without changing the composition of the solutionmentioned in step (d1) between consecutive additions. The gradient isformed by the dissolving of the composite of step (c) or (c1) by thesolution of step (d1). Using this procedure, one obtains a gradientlayer (transition region) of between 10 and 1000 micrometers in depth.

Depending on the volume of solution applied in step (d1), the depth oftransition region can be adjusted. It can generally be less than 1, 10,50, 70, 90, 100, 150, 190, 200, 210, 250, 300 micrometers in depth, or avalue in the range between any of the two aforementioned depths,preferably 150 to 250 micrometers, and most preferably about 200micrometers.

The volume of solution applied in step (d1) may be less than 5, 10, 15,20 25, 30, 35, 40, 45 or 50 microlitres, or a value in the range betweenany of the two aforementioned volumes. It is preferably between 15 and35 microlitres, more preferably between 20 and 30 microlitres and mostpreferably about 25 microlitres.

According to one aspect of the invention, the solution of step (d1)comprises plasticiser, polymer and ionophore in solvent (e.g. THF). Thecomposition of the membrane components (plasticiser+polymer+ionophore),may comprise more than 0, 1, 10, 20, 30, 40, 45, 50, 55, 60, 65, 70, 75,70, 75 or 80% plasticiser or a value in the range between any of the twoaforementioned percentages, preferably 55 to 75%, more preferably 60 to70% and most preferably about 65% plasticiser. The plasticiser may beany of the art, including o-nitrophenyl octyl ether (o-NPOE), dioctylsebacate (DOS), bis(2-ethylhexyl)phtalate (DOP),tris(2-ethylhexyl)phosphate (TOP) or tris(2-ethylhexyl)trimellitate(TOTM), but is preferably DOS.

The composition of the membrane components(plasticiser+polymer+ionophore) in the solution of step (d1), maycomprise less than 20, 22, 24, 26, 28, 30, 32, 33, 34, 36, 38, 40, 42,44, 46, 50, 60, 70, 80, 90, 100% polymer or a value in the range betweenany of the two aforementioned percentages, preferably 28 to 38%, morepreferably 32 to 34% and most preferably about 33% polymer. The polymercomprises the same polymer used in steps (a) to (c) or (a1) to (c1)described above.

The composition of the membrane components(plasticiser+polymer+ionophore) in the solution of step (d1), maycomprise less than 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7,8, 9 or 10% ionophore or a value in the range between any of the twoaforementioned percentages, preferably 0.1 to 3%, more preferably 0.5 to2.5% and most preferably 1 to 2% ionophore. The ionophore may be any ofthe art, depending on the desired application, includingtetra(p-chloro)phenylborate (TCPB) for cationic analytes, ormethyltridodecylammoniumchloride (MTDDACl) for anionic analytes or anyof those listed in FIG. 11.

The proportion of solvent (e.g. THF) in the solution of step (d1) may beone part membrane components (plasticiser+polymer+ionophore) to lessthan 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24,25, 26, 27, 28, 29, 30, 40, 50, 60 parts solvent, or a value in therange between any of the two aforementioned values. The preferred ratio(w/v) of membrane components (plasticiser+polymer+ionophore) to solventis 1:(8 to 12), more preferably 1:(9 to 11), most preferably about 1:10.

The composition of membrane components (plasticiser+polymer+ionophore)may vary within the above mentioned limits. The skilled person would usehis normal judgment to prepare a composition, where necessary adjustingthe relative proportions of membrane components to obtained the desiredcomposition.

According to one aspect of the invention, the solution of step (d1)comprises polymer and ionophore in solvent (e.g. THF), and noplasticiser. The composition of the membrane components (saidpolymer+said ionophore), may comprise less than or equal to 70, 75, 80,85, 90, 95, 96, 97, 98, 99% polymer or a value in the range between anyof the two aforementioned percentages, preferably 85 to 99%, morepreferably 90 to 99% and most preferably less than or equal to 98%polymer. The polymer is any having a Tg (glass transition temperature)below room temperature e.g. polysiloxane or preferablypoly(butylacrylate).

The composition of the membrane components (polymer+ionophore) in thesolution of step (d1), may comprise less than 0.1, 0.25, 0.5, 0.75, 1,1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9 or 10% ionophore or a value in therange between any of the two aforementioned percentages, preferably 0.1to 3%, more preferably 0.5 to 2.5% and most preferably 1 to 2%ionophore. The ionophore may be any of the art, depending on the desiredapplication, including tetra(p-chloro)phenylborate (TCPB) for cationicanalytes, or methyltridodecylammoniumchloride (MTDDACl) for anionicanalytes.

The composition of membrane components (polymer+ionophore) may varywithin the above mentioned limits. The skilled person would use hisnormal judgment to prepare a composition, where necessary adjusting therelative proportions of membrane components to obtained the desiredcomposition.

It is an aspect of the invention that the polymer used in steps (a) or(a1) and (d) or (d1) (and (f)) when plasticiser is absent, is the same,and is any having a Tg (glass transition temperature) below roomtemperature e.g. poly(butylacrylate) or polysiloxane. It is also anaspect of the invention that the polymers used in steps (a) or (a1) and(d) or (d1) and (f) when plasticiser is absent, are different. Thepolymer of steps (a) or (a1) may be any of poly(n-butylacrylate),polycarbonate, polystyrene, polymethylmethacrylate,poly(vinylchloride-co-vinylacetate-co-vinylalcohol) and preferablypolyvinyl chloride (PVC), the polymer of steps (d) or (d1) and (f) maybe poly(butylacrylate), polysiloxane or any polymer having a Tg (glasstransition temperature) below room temperature.

In this embodiment the proportion of solvent in the solution of step(d1) may be one part membrane components (polymer+ionophore) to lessthan 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24,25, 26, 27, 28, 29, 30, 40, 50, 60 parts solvent, or a value in therange between any of the two aforementioned values. The preferred ratio(w/v) of membrane components (polymer+ionophore) to solvent is 1:(8 to12), more preferably 1:(9 to 11), most preferably about 1:10.

The number of repetitions performed in step (f) and (f1) can veryaccording to the desired gradient profile, transition depth and volumeof solution applied in step (d) or (d1). The number of repetitions maybe less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 or a value in the range between any of the two aforementionedvalues. The preferred number of repetitions may be between 1 and 10,more preferably between 1 and 7 and most preferably between 1 and 3.

After hydration, the electrode is ready to be used.

According to one aspect of the invention, the analyte is present as anion pair in the electrode, particularly in the membrane region. In orderto obtain this ion pair, the following procedures may be used:

-   -   The ion pair (or analyte) may introduced by adding it to the        membrane components during fabrication (e.g. adding to the        solution of step (c) or (c1).    -   The ion pair may formed by direct contact of the finished        electrode with a solution of the analyte (e.g. 30 mg dapoxetine        in a 900 ml solution of 0.1M HCl). During this process, the        analyte is extracted from the solution into the electrode, and        the ion pair is formed in situ. The process may take up to 5        hours.

In another embodiment of the present invention, an electrode comprisinga polysiloxane membrane is constructed as follows:

a3) preparing a paste comprising:

-   -   a suspension of electrically conducting particles used in        step (a) or step (a1) above, with or without solvent, or    -   a suspension of electrically conducting particles used in        step (a) or step (a1) above, with or without solvent, in which        the polymer is polysiloxane, or    -   a suspension of electrically conducting particles used in        step (a) or step (a1) above, with or without solvent, in which        the polymer is DC 200 silicon oil,        b3) inserting an electrical conductor therein,        c3) adding to a surface of the particle/polymer composite a        mixture comprising base, curing agent, ionophore, optionally        dissolved in solvent (e.g. THF),        d3) degassing the construction,        e3) heating the construction, and        f3) obtaining a potentiometric electrode with composite gradient        properties and no interfaces. After hydration one obtains a        working electrode.

Base and curing agents are components known in the art and may be anysuitable for use in polymerisation. They are preferably components ofSylgard 184 (Dow Corning)

In this embodiment the proportion of optional solvent (e.g. THF) in thesolution of step (c3) may be one part solid mixture to less than 0.1,0.2, 0.3, 0.4, 0.5, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 40,50, 60 parts THF, or a value in the range between any of the twoaforementioned values. The preferred ratio (w/v) of solid components tosolvent is 1:(0.1 to 1), most preferably about 1:0.5.

The construction may be heating in step e3) to an temperature equal toor greater than 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 210, 220, 230, 240, 250 deg C., or a value in therange between any of the two aforementioned temperatures. Preferably,the temperature is 100 to 250 deg C., more preferably, 130 to 220 degC., or most preferably 150 to 200 deg C.

The ionophore may be any of the art, depending on the desiredapplication, including tetra(p-chloro)phenylborate (TCPB) for cationicanalytes, or methyltridodecylammoniumchloride (MTDDACl) for anionicanalytes or any of those listed in FIG. 11.

The composition of the membrane components (base+curing agent+ionophore)in step (c3), may comprise less than 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2,2.5, 3, 4, 5, 6, 7, 8, 9 or 10% ionophore or a value in the rangebetween any of the two aforementioned percentages, preferably 0.1 to 3%,more preferably 0.5 to 2.5% and most preferably 1 to 2% ionophore.

Ion pairs can be introduced as described above or can be introduced byswelling in organic solvents containing the analyte. The preferredsolvents are THF or DOS.

Properties of the polysiloxane can be tuned by introduction of DOS orother plasticisers. Silicon used was Sylgard 184 DOW CORNING.

In the construction of gradient polymer comprising two ‘back to back’gradients described previously, any two of the above mentioned gradientpolymers or electrodes are joined together at the surface of themembrane region (or lowest concentration of electrically conducingparticles) by means of the solvent (e.g. THF, DMSO).

FIG. 1 depicts a cross-section of an example of a potentiometricelectrode 9, according to the invention. The electrode comprises ahousing (isolator) 5, polymeric material 10, 11 and an electricalconnection 2. Electrically conducting particles 3, are disposed in theconducing composite 10. Ionophores 4 are disposed in the membrane region11. The ionophores 4 are least concentrated towards the electricalconnection 10 exit end of the cylinder, and most concentrated towardsthe sample contact end 8 of the cylinder. The ionophores 4 and theelectrically conducting particles 3 mix in a transition region 12.

FIG. 2 depicts a cross-section of another example of a potentiometricelectrode 9, according to the invention. The electrode comprises ahousing (isolator) 5, cylinder made from polymeric material 1 and anelectrical connection 2. Electrically conducting particles 3, aredisposed in a gradient 6 through the polymeric material 1. Theelectrically conducting particles 3 are most concentrated towards theelectrical connection 2 exit end of the polymeric material 1, and leastconcentrated towards the sample contact end 8 of the polymeric material.Ionophores 4 are disposed in a gradient 7 through the polymericmaterial. The ionophores 4 are least concentrated towards the electricalconnection-exit end of the cylinder 1, and most concentrated towards thesample contact end 8 of the polymeric material. The ionophores 4 and theelectrically conducting particles 3 mix in a transition region.

FIGS. 10A and 10B depict an example of a flow cell 17 according to theinvention. The outlet of an HPLC column 18 connects to an inlet 19 tothe flow cell 17. One or more spraying devices 15 provide a spray ofeluent 16, which is directed over the sample contact surface 8 of theelectrode 9 of the present invention. A reference electrode 13 is alsopresent in the flow cell. FIG. 10B illustrates the simple removal andinsertion of the electrode 9 from the flow cell 17.

EXAMPLES

The invention is illustrated by means of the following non-limitingexamples

1a. Preparing an Electrode (A).

A composite material forming the substantial part of the electrode wasmade by preparing a suspension of graphite particles and PVC in THF(tetrahydrofuran). The composite material was provided onto a copperelectrode by evaporating the suspension thereon. After drying thecomposite material, the electrode was extended by the addition ofionophore-containing layers as follows.

Several mixtures were prepared, each containing a different ionophore.

-   -   A mixture containing compounds 1 to 4 as ionophore comprised 1%        compound, 0.2% methyltridodecylammonium chloride (MTDDACl), 33%        PVC and 65.8% plasticiser as the final layer contacting the        sample.    -   The mixtures based on compounds 5-8 and 10 comprised 1%        ionophore, 33% PVC, and 66% plasticiser as the final layer        contacting the sample.        -   A mixture incorporating compound 9 comprised 1% ionophore,            27% PVC and 72% plasticiser as the final layer contacting            the a sample.        -   A mixture based on MTDDACl as an ionophore contained 6%            MTDDACl, 33% PVC and 61% plasticiser as the final layer            contacting the sample.

The anion-sensitive electrodes were conditioned with the running eluentin the LC system until a stable baseline was obtained (few hours).

1b. Preparing an Electrode (Alternative B).

An electrode suitable for use in HPLC potentiometric sensing is made ofa PVC cylinder (1×5 cm) with a central hole of 3 mm diameter (see, forexample, FIG. 1). One side of the hole is sealed by a copper rod. Theother side of the hole is filled with conducting composite of 60%graphite and 40% PVC:

-   -   1) A suspension of 60% graphite and 40% high molecular weight        PVC in THF (1/3 ratio/solid THF) is prepared by mixing and        stirring.    -   2) The hole is filled with the suspension of step (1).    -   3) The suspension is dried by room or elevated temperature.    -   4) Step 2 and 3 are repeated until the hole is completely        filled.    -   5) The surplus of graphite/PVC composite is removed by grinding        and polishing.

The membrane and the gradient are prepared as described:

-   -   1) 65% DOS, 32% PVC and 2% TCPB are mixed in a 100 mg quantity.    -   2) This mixture is dissolved in 1 ml THF    -   3) One drop (25 ul) is deposed onto the cylinder.    -   4) THF is evaporated at room temperature (˜5 mm).    -   5) Step 3 and 4 are repeated at least three times (in total).

Note: the gradient is formed by dissolving of the composite (60%graphite 40% PVC) in the membrane/THF solution. Using the describedprocedure one obtains a ±200 um layer in which the concentration of DOSand ions gradually decreases and the concentration of PVC and graphitegradually increases. After hydration one obtains a working electrode.

Life Span in HPLC Applications

The life span of two electrodes was tested by continuous exposure to aflow of distilled water at a flow rate of 1 ml/mm. At regular intervalsthe quality of the electrodes was tested by HPLC experiments. Theseconsisted of the separation and potentiometric detection of 6 amines(methyl-, ethyl-, propyl-, butyl-, pentyl- and hexylamine) on auniversal cation column (4.6×100 Alltech) with a 1 mM HNO₃ 5%Acetonitrile eluent. After 4 months and 127 liters of distilled water,the quality of the electrodes was still acceptable for routinepotentiometric measurements. The studied time span is equivalent with 1year of use with a workload of 40 hours/week at a 1 ml/mm flow rate.

1c. Preparing an Electrode (Alternative C)

The electrodes for HPLC applications may be adapted to the specificneeds of the dissolution application.

The electrodes for use in dissolution applications are prepare asdescribed in Example 1b. The PVC cylinder is glued onto a hollow glasstube with a diameter of 3 mm. In order to protect the glue from thesamples, a silicon tube is positioned over the glass PVC transition.

In order to obtain reproducible measurements in batch (dissolution) theanalyte is present as ion pair in the electrode membrane. In order toobtain this ion pair there are three possible procedures:

1) Introduction of the ion pair by adding it to the membrane componentsduring fabrication (step 1 of membrane preparation).

2) Adding the analyte to the membrane mixture during step 1 or 2 ofmembrane preparation.

3) Post production formation of the ion pair: the ion pair is formed bydirect contact of the electrode with a solution of the analyte (e.g. 30mg/900 ml dapoxetune/0.1M HCl). During this process the analyte isextracted from the solution into the electrode, and the ion pair isformed in situ. This process takes typically up to 5 hours.

Life Span in Dissolution Applications

The experimental data enables us to deduce a live span of ˜6 months forthe electrodes in dissolution applications.

1d. Preparing an Electrode (Alternative D)

Alternative preparation of the composite. A uniform composite can beobtained by following method:

1) A suspension of 60% graphite and 40% high molecular weight PVC in THF(1/3 ratio solid/THF) is prepared by mixing and stirring.

2) The mixture is injected in distilled water and precipitates.

3) The precipitate is reduced in size by blending.

4) The precipitate is filtered and dried.

5) The dry residue is pressed into a pellet by a IR pellet press (3ton).

6) The composite pellet is fixed in a PVC tube and a copper wire isinserted.

Membrane deposition and gradient formation are identical to the previousmethods (Examples 1b, 1c).

1e. Preparing an Electrode (Alternative E)

In the poly(butylacrylate) membrane electrode, PVC and plasticiser canbe replaced by poly(butylacrylate) in the construction of theelectrodes. The replacement can be partial i.e. PVC and DOS are replacedby poly(butylacrylate) during membrane and gradient construction), orcomplete i.e. all PVC (except the isolator) is replaced bypoly(butylacrylate).

In the first case the electrode construction is 60% graphite, 40%PVC/gradient/98% poly(butylacrylate), 2% ions and ionophores

In the second case the electrode consists of 60% graphite, 40%poly(butylacrylate)/gradient/poly(butylacrylate), 2% ions and ionophores

Poly(butylacrylate) composite can be used as base for nonpoly(butylacrylate) based membranes, e.g. PVC/DOS. In this case thecomposition of the electrode is: 60% graphite, 40%poly(butylacrylate)/gradient/65% DOS, 32% PVC, 2% ions and ionophores.

Note: The 60% graphite 40% poly(butylacrylate) composite is an idealself gluing base for mounting of any polymer based membrane.

Poly(n-butylacrylate) can be replaced by cross linkedpoly(butylacrylate), cross linking is initiated after mixing allcomponents and after creation of the gradient.

1f. Preparing an Electrode (Alternative F)

The polysiloxane membrane electrode is constructed as follows:

1) In a hollow glass tube a paste is introduced in which an electricalconductor (copper wire) is fitted as schematically depicted in FIG. 1.

This paste consists of:

60% graphite (1-2 micron) 30% PVC, or

60% graphite (1-2 micron) 30% polysiloxane, or

60% graphite (1-2 micron) 30% DC 200 silicon oil,

2) On this layer polysiloxane is applied as described:

Base and curing agent are mixed and ˜2% TCPB and/or ionophore are added.In order to become a more extended gradient this mixture can bedissolved in THF. Typically a 2/1 ratio mixture/THF is applied.

The mixture (with our without THF) is applied onto the paste of step 1).The construction is degassed by vacuum. The electrode is heated to150-200 deg C.; the temperature depends on the ratio base/curing agent.After hydration one obtains a working electrode.

Ion pairs can be introduced as described above (Example 1c) or can beintroduced by swelling in organic solvents containing the analyte. Thepreferred solvents are THF or DOS.

Properties of the polysiloxane can be tuned by introduction of DOS orother plasticisers. Silicon used was Sylgard 184 DOW CORNING.

1g. Preparing an Electrode (Alternative G)

1) A suspension of 60% graphite and 40% high molecular weight PVC in THF(113 ratio solid/THF) is prepared by mixing and stirring.

2) The mixture is injected in distilled water and precipitates.

3) The precipitate is reduced in size by blending.

4) The precipitate is filtered and dried.

5) The dry residue is pressed into a pellet by a IR pellet press (3ton).

6) The composite pellet is fixed in a PVC tube and a copper wire isinserted.

7) 65% DOS, 32% PVC and 2% TCPB are mixed in a 100 mg quantity.

8) This mixture is dissolved in 1 ml THF

9) One drop (25 ul) is deposed onto the cylinder.

10) THF is evaporated at room temperature (˜5 nin).

11) Step 3 and 4 are repeated at least three times (in total).

12) Make a second component by repeating step 1-11

13) Glue both membrane sides of the gradient polymer together by meansof solvent (THF).

2. Synthesis of the Compounds 1 to 10.

Compounds 1 and 2:

Tris(2-aminoethyl)amine was mixed with 3 equiv. of octadecyl isocyanate,or phenyl isocyanate in chloroform solution at room temperature. Aftervigorous exothermic reaction, the mixtures were heated at reflux for 3hours, cooled to room temperature, filtered off, washed with chloroformand dried.

Yield of 1: 87%, 2-93%. LSIMS (NBA): 1-1032 (M+H)⁺, 2-504 (M+H)⁺

Compound 3:

2,6-Diaminopyridine was dissolved in warm cyclohexane to obtain a clearsolution. To this hot solution one equivalent of octadecyl isocyanatewas added slowly. The mono substituted product precipitated during thereaction course, preventing the product from double substitution. Aftercooling, the white product was filtered off, washed with cyclohexane anddried at reduced pressure.

Yield quantitative. LSIMS (NBA): 405 (M+H)⁺

Compound 4:

2,6-Diaminopyridine was dissolved in warm chloroform to obtain a clearsolution. To this hot solution, two equivalents of octadecyl isocyanatewere added slowly. The disubstituted product precipitated during thereaction course. After cooling, a double amount of hexane was added toprecipitate the compound. The white product was filtered off, washedwith hexane and dried at reduced pressure.

Yield 95%. LSIMS (NBA): 700 (M+H)⁺

Compound 5:

The hexaaza compound with secondary amino functions was synthesized asdescribed in Pauwels, T. F.; Lippens, W.; Herman, G. G.; Goeminne, A. M.Polyhedron 1998, 17, 1715-23, and Menif, R.; Martell, A. E. Journal ofthe Chemical Society-Chemical Communications 1989, 1521-23. ItsN-hexaoctyiderivative was obtained as follows: The solution of thehydrochloride salt of the hexa-aza compound (0.40 g, 0.62 mmol) andK₂CO₃ (5 g) in 200 ml dry acetonitrile was stirred for 24 h undernitrogen blanket. A solution of n-octylbromide (0.78 g, 4 mmol) in 50 mlof dry acetonitrile was added dropwise to the stirred mixture. Acatalytic amount of tetrabutylammonium iodide (0.15 g) was added and themixture was refluxed for two days. The warm mixture was filtered. Thesolvent was removed in vacuo to give a brownish oil to which 100 ml ofsodium hydroxide solution (pH=12) and 100 ml of chloroform were added.The water phase was extracted several times with additional portions of100 ml of chloroform. The chloroform layers were collected, dried overanhydrous K₂CO₃ and evaporated under reduced pressure. The resulting oilwas purified by column chromatography on silica gel withchloroform-ethanol (80:20) as eluent, which gave a yellow oil afterevaporating the eluent (201 mg, 30% yield).

1H-NMR (CDCl₃): δ 0.81 (t, 18H), 1.18-1.36 (m, 72H), 2.28-2.39 (m, 28H),3.36 (s, 8H), 7.07-7.13 (m, 8H).

Compounds 6 and 7:

The synthesis of these compounds was described in Jarzebinska, A.;Pietraszkiewicz, M.; Bilewicz, R. Materials Science & EngineeringC-Biomimetic and Supramolecular Systems 2001, 18, 61-64. Compounds 8, 9and 10 were synthesized under an argon atmosphere.

Compound 8:

The starting hexaamine (structure as for compound 6 where R═R1=H, FIG.11) was synthesized according to the procedure given by Pietraszkiewicz,M.; Gasiorowski, R. Chemische Berichte 1990, 123, 405-06. The solutionof hexaamine (1 equiv.) and acrylonitrile (12 equiv.) was stirred for 6h at 40° C. Evaporation yielded white solid. Then, the cyanide groupswere reduced with LiAlH₄ (6 equiv.) in THF at reflux under Ar. After 5hours, the excess of LiAlH₄ was quenched with 10% aqueous solution ofNaOH. The isolated and dried compound was mixed with octadecylisocyanate (6 equiv.) in dry THF at elevated temperature. The stirringwas continued for 3 hours, then the solvent was evaporated. MeOH wasadded, and the formed solid was filtered off, washed with methanol, anddried in vacuum.

MALDI-TOF MS: 2547 (M⁺).

Compound 9:

To the first generation of dendrimer based on a macrocyclic hexaamine(its synthesis was described in Pietraszkiewicz, M.; Gasiorowski, R.Chemische Berichte 1990, 123, 405-06), octadecyl isocyanate (12 equiv.)was added. The mixture was stirred continuously for 48 h at elevatedtemperature. The brown solid thus obtained was washed three times withwarm EtOH. After cooling, the solid precipitated from the solvent. Next,the product was filtered and dried in vacuum.

MALDI-TOF MS: 5138.0 (M⁺).

Compound 10:

To the rapidly stirred mixture of 4 g hexaamine (mentioned above) in THFat reflux, succinic anhydride (1 g) was added. Reflux and stirring wascontinued for 2 h. The succinic amido-acid derivative was filtered off,washed with cold THF and dried. The solid was reduced in this same wayas described above for compound 8. After reduction, the solid was mixedwith 30% aqueous formaldehyde (40 ml) and formic acid (30 ml) and heatedfor 6 h at reflux. Water and acid were evaporated and the product wasdried in vacuum. In the next step the compound was dissolved in THF andstirred with octadecyl isocyanate (1 equiv.) for 6 h at elevatedtemperature. After evaporation of THF the solid was dried in vacuum.MALDI-TOF MS: 904.6 (M⁺); MS (ES⁺): 904.7 (M⁺)

4. Instrumentation

Liquid chromatography was performed using a Spectra Physics 8810isocratic pump, a Valco injector (10 μL loop), column 125×4 mm filledwith reversed-phase Lichrospher 100-5 RP-8, (Merck), eluent 1 mM H₃PO₄.The flow-rate was 0.5 mL min⁻¹. The column outlet was directedperpendicularly towards the sensitive membrane of the coated-wireelectrode in a flow-cell (Zielinska, D.; Poels, I.; Pietraszkiewicz, M.;Radecki, J.; Geise, H. J.; Nagels, L. J. Journal of Chromatography A2001, 915, 25-33). The distance from the LC tubing outlet to theelectrode was 100 μm. The membrane potential was measured against anOrion 800500 Ross® reference electrode using a high impedance (10¹³Ω)amplifier. The detection signals were recorded on a PC 1000 dataacquisition system from Thermo Separation Products.

5. Potentiometric Detection of Organic Acids.

A set of 7 (mostly dicarboxylic) acids were used as test substances.These acids are widely found in nature, and in the food/beverageindustry. These acids are quite hydrophilic, with log P values mostlylower than (−1) (see Table 1). Membranes, whose components displaylipophilic character, have the tendency to be unsensitive to hydrophilicanalytes. The choice of the ionophore host molecules is essential.Molecules forming the strongest complex with the ionophore are detectedmost sensitively.

FIG. 12: Upper tracing: Separation of the organic acids on aLichrosphere 100-5 RP8 column (Merck), 125×4 mm, eluent 1 mM H₃PO₄ at0.5 mL min⁻¹ flow-rate. 10 μL injection of a mixture of tartaric- (1,1×10⁻⁵M), malonic- (2, 2×10⁻⁷M), malic- (3, 4×10⁻⁶M), lactic- (4,1×10⁻⁴M), citric- (5, 1×10⁻⁵M), fumaric- (6, 2×10⁻⁵M), succinic acid (7,2×10⁻⁵M). Lower tracing: Same conditions as upper tracing, injection ofcommercial lager pills beer after filtration on a 0.45 μm filter. Theelectrode coating used contained 33.8% PVC, 66% o-NPOE, 0.2% MTDDACl,and 1% ligand 3.

All components were detected very sensitively, the highest sensitivitybeing obtained for malonic acid: peak nr. 2 is due to 120 pg. Thedetection limit for this acid is in the low picogram range. The lowertracing presents a chromatogram of an injected beer sample (lager pills,no cleanup except filtration over a 0.45 μm filter, no dilution, 10 μLinjection) in the same conditions. Tartaric- (1) and lactic- (4) acidcould be identified.

FIG. 13 shows a HPLC chromatogram recorded using a coated-wire electrodeincorporating ligand 9 and TOTM. The compound 9, of dendrimeric type, istightly packed with urea units. Injected concentration: 1×10⁻⁵M, peakidentification: (1) tartaric acid, (2) malonic acid, (3) malic acid, (4)lactic acid, (5) citric acid, (6) fumaric acid, (7) succinic acid.Electrodes based on compound 9 were less sensitive but showed preferencefor citric acid (peak 5 in the chromatogram of FIG. 13).

FIG. 14 shows chromatograms obtained using a coated-wire electrodeincorporating ligand 10 and TOP as plasticiser. In case of the compound10, there is only one carbamoyl group available. Trace A) Injectedconcentration: 1×10⁻⁵ M, peak identification: (1) tartaric acid, (2)malonic acid, (3) malic acid, (4) citric acid, (5) fumaric acid, (6)succinic acid. Trace B) Injected concentration and peak identification:(1) 5×10⁻⁴ M tartaric acid, (2) 2×10⁻⁴M malonic acid, (3) 2×10⁻⁴ M malicacid, (4) 5×10⁻³ M lactic acid, (5) 5×10⁻⁴ M citric acid, (6) 5×10⁻⁶ Mfumaric acid, (7) 5×10⁻⁴ M succinic acid. Electrodes based on thiscompound are very sensitive for fumaric acid (FIG. 14).

Table 1 lists detection limits (in pmol injected) of the 7 organicacids, obtained with the chromatographic system described in Example 4,with different ionophores in the potentiometric detector. TABLE 1detection limits (in pmol injected) of electrodes for 7 organic acids.Minimum amount of acid (pmol) required for detection in electrodescomprising compound (cmpd) numbers below Acid Log P Cmpd 1 Cmpd 2 Cmpd 3Cmpd 4 Cmpd 5 Cmpd 6 MTDDACI Tartaric −1.39 20 12 11 23 55 20 200Malonic −0.34 1.6 1.0 1.1 1.6 4 4 200 Malic −1.22 6.5 4.0 4.2 5.6 20 15200 Lactic −1.31 830 910 650 1200 5000 1900 1000 Citric −1.65 23 10 7 1510 50 200 Fumaric +0.49 280 140 60 310 650 80 300 Succinic −1.11 77 5647 86 330 200 700

Comparing the data obtained for electrodes incorporating the quaternarysalt MTDDACl, where only electrostatic interactions occur, it is clearthat extra interactions originating from compounds, makes the membranesmuch more sensitive.

Thus for malonic acid detected on membranes modified by open chain urea(1-4) and macrocyclic compound 8 the detection limit is down to 1 pg(see Table 1). This is two orders of magnitude lower than for MTDDAClbased electrodes. All other acids were also detected sensitively, atinjected concentrations varying from 4×10⁻⁴ to 2×10⁻⁵ M.

With hexaamine ionophores possessing alkyl chains, (compounds 5 to 7,FIG. 11), detection limits are drastically reduced compared withMTDDACl-based electrodes for 4 of the 7 acids (tartaric, malonic, malic,citric), with the most drastic reduction for malonic acid. All theazamacrocycles with lipophilic alkyl chains display very similarselectivities and detection limits, regardless the size of themacrocyclic cavity (compounds 5 to 7).

Malonic acid, as monoanion, can interact with ionophoreselectrostatically and by hydrogen bonding (Menif, R.; Martell, A. E.Journal of the Chemical Society-Chemical Communications 1989, 1521-23).Moreover, this acid is less hydrophilic (except of fumaric acid) thanother analytes.

The best results for compound 10 were observed for fumaric acid (seeFIG. 14), but the level of detection of other acids was worse than thatfor MTDDACl. The compound 10 possesses only one large lipophilic tailwith a functional group. Probably, the macrocyclic unit does not lie atthe membrane-water interface. This could lead to diminishedelectrostatic interactions of the acids with the compound at theinterface.

Generally, detection limits were in the order malonic- (bestdetected)<citric- malic- and tartaric-<fumaric- and succinic-<lacticacid. For the most sensitively detected acid, malonic acid, detectionlimits as low as 1 pmol (injected amount) are obtained. This is quitesensitive, but is still a factor of 100 above the detection limitsobtainable with HPLC/potentiometric detection for basic drugs with highlog P values (e.g. bromhexine, ambroxol and clenbuterol with log Pvalues of 4.03, 3.24 and 2.91 respectively).

6. Calibration curves

FIG. 15 shows calibration curves obtained for three acids, with anelectrode comprising the octaazamacrocycle compound (compound 7). Inbatch techniques, potentiometric detectors are always used in that partof the calibration curve where the signal (mV) is linearly related tolog(C_(analyte)). The calibration curve from FIG. 15 shows a linearrelationship between the signal (peak height or area) and the analyteconcentration.

The correlation is high, i.e. p=0.99999 (n=5) for malonic acid and atleast 0.97 (n=4) for all other acids listed in Table 1 (not all data areplotted in FIG. 15). When used in HPLC conditions, the classicalmethodology of potentiometry has to be somewhat adapted to thesituation. With a potentiometric sensor which continuously monitors thesignal, one can work in the lower part of the calibration curve. It hasbeen shown theoretically (De Backer, B. L.; Nagels, L. J. AnalyticalChemistry 1996, 68, 4441-45), that in this part of the calibrationcurve, a linear relationship should be expected between the injectedconcentration, and the peak area (or peak height). For all acids fromTable 1, this linearity was obtained in the 1 to 100 μM region.

7. Long-Term Stability of the Electrodes

Electrodes containing the azamacrocyclic compounds 5 and 7 had anoperational lifetime (continuous use in chromatographic conditions) ofat least 3 months. Their calculated high log P values were 20.6 and16.7, respectively. Electrodes containing azamacrocyclic compound 6 witha significantly lower logP of 9.18 had a reduced operational lifetime ofa few weeks. The minimal lipophilicity log P, required forionophore-containing electrodes with a lifetime of 30×24 (days)×(h) uponexposure to an aqueous solution is generally estimated to be around 10.Electrodes containing the open chain urea compounds 1 to 4 also hadoperational lifetimes in the order of 2 weeks. The log P values ofcompounds 1 to 4 are 25.4, 10.1, 9.6 and 18.6 respectively.

10 synthetic ligands were tested and compared as ionophores in electrodemembranes which are responsive to carboxylic acids. All these ligandslargely improved (in comparison to MTDDACl) detection limits for theHPLC determination of the di- and tricarboxylic acids. The highestsensitivities were obtained for the open-chain urea compound 3. Compound7 combines high sensitivity with long operational lifetime.

8. Application in “Dissolution Testing” of Pharmaceutical Drugs.

Determining the rate of dissolution of an active pharmaceuticalingredient (API) from a dosage form is an important quality indicatingtest in the pharmaceutical industry. Potentiometric sensors can be veryuseful in this respect. Classical UV detection systems allow only a verylimited concentration range to be measured as the useful linear dynamicrange of a UV detector is limited (0-2 absorbance units). The outputsignal of a UV detector gets saturated because the detector's responseis linearly related to the concentration of the dissolved substance.Potentiometric sensors have the advantage that their response isdependent on the logarithm of the concentration. This means that if onegets good signals at the low concentration side, even very highconcentrations can be monitored without saturating the sensor outputvoltage. Such behaviour can be seen in FIG. 16. Moreover, potentiometricsensors do not suffer from factors limiting the use of spectroscopicmethods such as effects of flow cell dimensions, turbidity, and thepresence of undissolved particles and of air bubbles.

Dissolution of 8 mg galantamine (spherical particles in capsules marked“reminyl CR”) in 100 ml 0.001 M HCl was monitored by a potentiometricelectrode of the present invention. The HCl solution was stirredcontinuously with a magnetic stirring bar. In FIG. 17, curve B shows theoutput signal of the potentiometric sensor. The signal was monitoredversus a reference electrode which was also hanging in the HCl solution.Curve A was calculated from curve B using the smoothed data from FIG.16. It shows the concentration versus time behavior of the basic druggalantamine. The electrode used contained PVC,tetra(p-chloro)phenylborate (TCPB) as an ionophore, and DOS as aplasticiser.

The observed response profile gives a good description of thedissolution behaviour. The potentiometric electrode of the inventionalso allows measurements to be done in media which damage the coatedwire type electrodes (e.g. media containing detergents).

1. A potentiometric electrode for selective analyte detection in a sample comprising: a sensing body made from polymeric material comprising: electrically conducting particles, which increase in concentration away from a sample contact surface, ionophore molecules, which increase in concentration towards the sample contact surface, and an electrical connection which passes proximal to said electrically conducting particles, wherein the ionophore molecules and the electrically conducting particles mix predominantly in a transition region, which depth is between 50 and 1000 micrometres.
 2. The potentiometric electrode according to claim 1 wherein the polymeric material comprises one or more of poly(n-butylacrylate), poly(butylacrylate), cross-linked poly(butylacrylate), polycarbonate, polystyrene, polymethylmethacrylate, poly(vinylchloride-co-vinylacetate-co-vinylalcohol), polysiloxane, DC 200 silicon oil, polyvinyl chloride, or high molecular weight polyvinyl chloride.
 3. The potentiometric electrode according to claim 1 wherein said electrically conducting particles are selected from the group consisting of carbon powder, graphite powder, synthetic graphite powder and combinations thereof and wherein said conducting particles have a diameter of 1 to 2 micrometer.
 4. The potentiometric electrode according to claim 1, wherein said electrically conducting particles are selected from the group consisting of electropolymerised materials, oxidized polypyrole and its derivatives, oxidized polythiophenes, polyaniline, noble metals, gold, platinum and combinations thereof.
 5. The potentiometric electrode according to claim 1, wherein said ionophore molecules are one or more selected from the group consisting of the compounds 1 to 10 in FIG.
 11. 6. The potentiometric electrode according to claim 1 wherein said ionophore molecules are one or more selected from the group consisting of tetra(p-chloro)phenylborate, methyltridodecylammoniumchloride and compounds 11 to 21 in FIG.
 11. 7. The potentiometric electrode according to claim 1 wherein the polymeric material is at least partly enclosed in a housing.
 8. A potentiometric electrode according to claim 1 wherein the polymeric material comprises plasticiser and wherein the plasticiser is more disposed in a region of polymeric material comprising ionophore molecules, than the remainder of the polymeric material.
 9. A method for making a potentiometric electrode according to claim 1 comprising the steps of: (a) preparing a suspension of electrically conducting particles in a solution comprising polymeric material or polymer in a solvent, (b) inserting an electrical conductor into the suspension, (c) drying the suspension, so forming a solid composite polymer with electrically conducting particles therein, (d) adding, to a surface of the composite of step (c) a solution comprising one or more of polymer, polymeric material, electrically conducting particles and ionophore molecules, (e) drying the mixture, (f) repeating steps (d) and (e), in such a manner that the transition region has a depth between 50 and 1000 micrometres, and (g) obtaining a potentiometric electrode.
 10. The method for making a potentiometric electrode according to claim 9, wherein the solution of step (a) comprises the polymer in a solvent, and wherein the solution of step (d) comprises polymer and ionophore, whereby a potentiometric electrode is obtained with composite gradient properties and no interfaces.
 11. The method for making a potentiometric electrode according to claim 9 wherein the solution of step (a) comprises the polymer in a solvent, and further comprising the steps of: (b1) injecting the suspension into distilled water, so forming a precipitate, (b2) reducing the size of the precipitate to form a residue, (c1) drying the residue by pressing to form a conducting particle/polymer composite, followed by steps (d) to (g) as defined in claim
 9. 12. The method according to claim 9 wherein the relative proportion (w/w) of said electrically conducting particles to said polymer is in the range 20 to 90% electrically conducting particles.
 13. The method according to claim 9 wherein said electrically conducting particles are selected from the group consisting of carbon powder, graphite powder, synthetic graphite powder and combinations thereof and wherein said conducting particles have a diameter of 1 to 2 micrometer.
 14. The method according to claim 9 wherein said electrically conducting particles are selected from the group consisting of polyaniline, noble metals, gold platinum, electropolymerised materials, oxidized polypyrole and its derivatives, oxidized polythiophenes and combinations thereof.
 15. The method according to claim 9 wherein said polymer is selected from the group consisting of poly(n-butylacrylate), poly(butylacrylate), polycarbonate, polystyrene, polymethylmethacrylate, poly(vinylchloride-co-vinylacetate-co-vinylalcohol), polysiloxane, DC 200 silicone oil, high molecule weight polyvinyl chloride (PVC) and combinations thereof.
 16. The method according to claim 9 wherein the proportion of the mass of solid components (electrically conducting particles and polymer), to the volume of solvent in the suspension of step (a) is 1:(1 to 10).
 17. The method according to claim 9 wherein the solvent of step (a) is selected from the group consisting of DMSO, 111 trichloro ethane, CCl₄, N,N-dimethylformamide and N,N-dimethylacetamide, THF and combinations thereof.
 18. The method according to claim 9 wherein the solvent of step (d) is selected from the group consisting of DMSO, 111 trichloro ethane, CCl₄, N,N-dimethylformamide and N,N-dimethylacetamide, THF and combinations thereof.
 19. The method according to claim 9 wherein said ionophore molecules are one or more selected from the group consisting of compounds 1 to 10 in FIG.
 11. 20. The method according to claim 9 wherein said ionophore molecules are one or more selected from the group consisting of tetra(p-chloro)phenylborate, methyltridodecylammoniumchloride and the compounds 11 to 21 in FIG.
 21. The method according to claim 9 wherein said polymer in step (d) is any which is in the rubber phase at or below room temperature.
 22. The method according to claim 21 wherein said polymer in step (d) is poly(butylacrylate).
 23. The method according to claim 21 wherein the composition of the solid components (polymer and ionophore) in step (d) comprises 85 to 99% polymer.
 24. The method according to claim 21 wherein the composition of the solid components (polymer and ionophore) in step (d), comprises 0.1 to 3% ionophore.
 25. The method according to claim 22 wherein the ratio (w/v) of solid components (polymer and ionophore) to solvent is 1:(8 to 12) in step (d).
 26. The method according to claim 9 wherein the solution of step (d) further comprises plasticiser.
 27. The method according to claim 26 wherein the composition of the membrane components (plasticiser, polymer and ionophore) in step (d) comprises 55 to 75% plasticiser.
 28. The method according to claim 26 wherein said plasticiser is selected from the group consisting of o-nitrophenyl octyl ether, dioctyl sebacate, bis(2-ethylhexyl)phtalate, tris(2-ethylhexyl)phosphate, tris(2-ethylhexyl)trimellitate and combinations thereof.
 29. The method according to claim 26 wherein the composition of the membrane components (plasticiser, polymer and ionophore) in step (d), comprise 28 to 38% polymer.
 30. The method according to claim 26 wherein the composition of the membrane components (plasticiser, polymer and ionophore) in step (d), comprises 0.1 to 3% ionophore.
 31. The method according to claim 26 wherein the ratio of the mass of membrane components (plasticiser, polymer and ionophore) to the volume of solvent is 1:(8 to 12) in step (d).
 32. A method for preparing a potentiometric electrode according to claim 1 comprising the steps of a3) preparing a paste comprising: a suspension of electrically conducting particles comprising polymeric material or a polymer, with or without solvent, or a suspension of electrically conducting particles comprising a polymer, with or without solvent, in which the polymer is polysiloxane, or a suspension of electrically conducting particles comprising a polymer, with or without solvent, in which the polymer is DC 200 silicon oil, b3) inserting an electrical conductor therein, c3) adding to a surface of the particle/polymer composite a mixture comprising base, curing agent, and ionophore, optionally dissolved in solvent, d3) degassing the construction, e3) heating the construction, and f3) obtaining a potentiometric electrode with composite gradient properties and no interfaces.
 33. An electrode obtainable using a method according to claim 9 or
 32. 34. A chromatographic flow cell comprising a potentiometric electrode according to claim 1 or
 33. 35. A chromatographic flow cell according to claim 34, comprising a nozzle allowing eluent to be sprayed onto a sample contact surface.
 36. An inline dissolution electrode comprising a potentiometric electrode according to claim 1 or
 33. 37. A gradient polymer substantially formed from polymeric material comprising two surfaces, said polymeric material comprising: electrically conducting particles which decrease in concentration away from one surface, ionophore molecules decrease in concentration away from the other surface, wherein the ionophore molecules and the electrically conducting particles mix predominantly in a transition region, which depth is between 50 and 1000 micrometres.
 38. A gradient polymer substantially formed from polymeric material comprising two surfaces, said polymeric material comprising electrically conducting particles, which decrease in concentration away from both surfaces.
 39. A gradient polymer according to claim 37 or 38, wherein the polymer comprises one or more of poly(n-butylacylate), poly(butylacrylate), cross-linked poly(butylacrylate), polycarbonate, polystyrene, polymethylmethacrylate, poly(vinylchloride-co-vinylacetate-co-vinylalcohol), polysiloxane, DC 200 silicon oil, polyvinyl chloride, or high molecular weight polyvinyl chloride.
 40. A gradient polymer according to claim 37 or 38 wherein the polymer comprises one or more electrically conducting particles selected from carbon powder, graphite powder, synthetic graphite powder electropolymerised materials, oxidized polypyrole and its derivatives, oxidized polythiophenes, polyaniline, noble metals, gold, platinum and combinations thereof.
 41. A gradient polymer according to claim 37 or 38, wherein the polymer comprises one or more of the ionophores selected from the group consisting of electropolymerised materials, oxidized polypyrole and its derivatives, oxidized polythiophenes, polyaniline, noble metals, gold, platinum and combinations thereof.
 42. A method for preparing a gradient polymer according to claim 37 or 38 comprising the steps of (a) preparing a suspension of electrically conducting particles in a solution comprising polymeric material or polymer in a solvent, (b) inserting an electrical conductor into the suspension, (c) drying the suspension, so forming a solid composite polymer with electrically conducting particles therein, (d) adding, to a surface of the composite of step (c) a solution comprising one or more of polymer, polymeric material, electrically conducting particles and ionophore molecules, (e) drying the mixture, (f) repeating steps (d) and (e), in such a manner that the transition region has a depth between 50 and 1000 micrometres, and (g) obtaining a potentiometric electrode. wherein one or more additional electrical conductors are incorporated in step (b).
 43. The method for preparing a gradient polymer according to claim 42, wherein the ionophore of step (d) is absent.
 44. A method for preparing a gradient polymer according to claim 42, further comprising the step of joining two gradient polymers together at the surfaces of lowest concentration of electrically conducting particles.
 45. A battery comprising gradient polymer according to claim 37 or
 38. 46. A variable resistor gradient polymer according to claim 37 or
 38. 47. A variable capacitor gradient polymer according to claim 37 or
 38. 48. A pressure sensor gradient polymer according to claim 37 or
 38. 49. A solvent or lipophilic molecule sensor gradient polymer according to claim 37 or 38
 50. A method for sensing analytes comprising measuring the response of the gradient polymer according to claim 37 or 38 to the analyte.
 51. A method for inline monitoring the dissolution of a drug from a drug formulation which comprises measuring the output signal of the potentiometric electrode of claim 1 thereby monitoring the dissolution of the drug.
 52. The method of claim 9, further comprising repeating steps (d) and (e) with decreasing concentrations of electrically conducting particles, and increasing concentrations of ionophore molecules. 